Method of inhibiting amyloid protein aggregation and imaging amyloid deposits using aminoindane derivatives

ABSTRACT

The present invention provides compounds of Formula (I) and a method of treating Alzheimer&#39;s disease using a compound of Formula (I), wherein: R 1  and R 2  include alkyl and phenylalkyl; R 3  is hydrogen or alkyl; and R 4  and R 5  include alkyl, alkoxy, carboxyl, alkoxycarbonyl, and nitro. Also provided is a method of inhibiting the aggregation of amyloid proteins using a compound of Formula (I) and a method of imaging amyloid deposits using compounds of Formula (I).

FIELD OF THE INVENTION

[0001] This invention relates to a method of inhibiting amyloid proteinaggregation and imaging amyloid deposits. More particularly, thisinvention relates to a method of inhibiting amyloid protein aggregationin order to treat Alzheimer's disease using aminoindane derivatives.

BACKGROUND OF THE INVENTION

[0002] Amyloidosis is a condition characterized by the accumulation ofvarious insoluble, fibrillar proteins in the tissues of a patient. Thefibrillar proteins that comprise the accumulations or deposits arecalled amyloid proteins. While the particular proteins or peptides foundin the deposits vary, the presence of fibrillar morphology and a largeamount of β-sheet secondary structure is common to many types ofamyloids. An amyloid deposit is formed by the aggregation of amyloidproteins, followed by the further combination of aggregates and/oramyloid proteins.

[0003] The presence of amyloid deposits has been shown in variousdiseases, each with its particular associated protein, such asMediterranean fever, Muckle-Wells syndrome, idiopathetic myeloma,amyloid polyneuropathy, amyloid cardiomyopathy, systemic senileamyloidosis, amyloid polyneuropathy, hereditary cerebral hemorrhage withamyloidosis, Alzheimer's disease, Down syndrome, Scrapie,Creutzfeldt-Jacob disease, Kuru, Gerstrnann-Straussler-Scheinkersyndrome, medullary carcinoma of the thyroid, Isolated atrial amyloid,β₂-microglobulin amyloid in dialysis patients, inclusion body myositis,β₂-amyloid deposits in muscle wasting disease, Sickle Cell Anemia,Parkinson's disease, and Islets of Langerhans diabetes type 2insulinoma.

[0004] A simple, noninvasive method for detecting and quantitatingamyloid deposits in a patient has been eagerly sought. Presently,detection of amyloid deposits involves histological analysis of biopsyor autopsy materials. Both methods have major drawbacks. For example, anautopsy can only be used for a postmortem diagnosis.

[0005] The direct imaging of amyloid deposits in vivo is difficult, asthe deposits have many of the same physical properties (i.e., densityand water content) as normal tissues. Attempts to image amyloid depositsdirectly using magnetic resonance imaging (MRI) and computer-assistedtomography (CAT) have been disappointing and have detected amyloiddeposits only under certain favorable conditions. In addition, effortsto label amyloid deposits with antibodies, serum amyloid P protein, orother probe molecules has provided some selectivity on the periphery oftissues, but has provided for poor imaging of tissue interiors.

[0006] Thus, it would be useful to have a noninvasive technique forimaging and quantitating amyloid deposits in a patient. In addition, itwould be useful to have compounds that inhibit the aggregation ofamyloid proteins to form amyloid deposits.

[0007] One of the most devastating diseases associated with amyloiddeposits is Alzheimer's disease. Alzheimer's disease is a degenerativebrain disorder characterized clinically by progressive loss of memory,cognition, reasoning, judgement, and emotional stability that graduallyleads to mental deterioration and ultimately death. Because Alzheimer'sdisease and related degenerative brain disorders are a major medicalissue for an increasingly aging population, the need for new treatmentsand methods for diagnosing the disorders are needed.

[0008] Several classes of compounds have been shown to have activityagainst Alzheimer's disease. The only two agents currently approved forclinical treatment of Alzheimer's disease are the acetylcholinesteraseinhibitors tacrine and donepezil (see U.S. Pat. No. 4,816,456). U.S.Pat. Nos. 5,716,975 and 5,523,314 relate to rhodanine derivatives usefulas hypoglycemic agents and for treating Alzheimer's disease.

[0009] The present invention provides a group of aminoindanyl analogsthat are inhibitors of amyloid aggregation and are thus useful fortreating Alzheimer's disease. The compounds are also useful as imagingagents because of their ability to selectively bind to amyloid proteins.

SUMMARY OF THE INVENTION

[0010] The present invention provides compounds of the Formula I

[0011] wherein:

[0012] R¹ and R² independently are hydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, (CH₂)_(n) phenyl or (CH₂)_(n) substituted phenyl,provided that one of R¹ and R² is other than hydrogen;

[0013] R⁴ and R⁵ independently arc hydrogen, halo, C₁-C₈ alkyl, C₂-C₈alkenyl, C₂-C₈ alkynyl, (CH₂)_(n) phenyl, (CH₂)_(n) substituted phenyl,NO₂, CN, CF₃, C₁-C₈ alkoxy, CO₂R⁶, tetrazolyl, NH(C₁-C₈ alkyl), N(C₁-C₈alkyl)₂, or SO₂R⁶;

[0014] R³ is hydrogen or C₁-C₈ alkyl;

[0015] R⁶ is hydrogen, C₁-C₈ alkyl, or (CH₂)_(n)phenyl or (CH₂)_(n)substituted phenyl;

[0016] n is an integer from 0 to 4 inclusive;

[0017] or a pharmaceutically acceptable salt, ester, amide, or prodrugthereof.

[0018] Preferred compounds have Formula I wherein one of R¹ and R² is(CH₂)_(n)phenyl or (CH₂)_(n) substituted phenyl, and R⁵ is CO₂R⁶,tetrazolyl, or SO₂ R⁶, and R⁶ is hydrogen.

[0019] A preferred group of compounds have Formula II

[0020] where R¹, R², R⁴, and R⁶ are as defined above.

[0021] Another preferred group of compounds have Formula III

[0022] where in R⁴, R⁵, and R⁶ are as defined above, and R⁷ is hydrogen,halo, NO₂, CN, C₁-C₈ alkyl, C₁-C₈ alkoxy, CF₃, NH₂, NH(C₁-C₈alkyl), orN(C₁-C₈alkyl)₂.

[0023] A further embodiment of this invention is a pharmaceuticalcomposition comprising a compound of Formula I together with a carrier,excipient, or diluent therefor.

[0024] Another embodiment of this invention is a method for inhibitingamyloid aggregation in a mammal comprising administering an effectiveamount of a compound of Formula I. A further method provided is a methodof treating Alzheimer's disease and central and/or peripheralamyloidosis syndromes in mammals comprising administering an effectiveamount of a compound of Formula I.

[0025] Also provided is a method of imaging amyloid deposits, the methodcomprising the steps of:

[0026] a. introducing into a patient a detectable quantity of a labeledcompound of Formula I;

[0027] b. allowing sufficient time for the labeled compound to becomeassociated with amyloid deposits; and

[0028] c. detecting the labeled compound associated with the amyloiddeposits.

[0029] In a preferred embodiment of the method, the patient has or issuspected to have Alzheimer's disease.

[0030] In another preferred embodiment, the labeled compound is aradiolabeled compound.

[0031] In another preferred embodiment, the labeled compound is detectedusing MRI.

[0032] Also provided is a pharmaceutical composition comprising acompound of Formula I.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The term “alkyl” means a straight or branched chain hydrocarbon.Representative examples of alkyl groups are methyl, ethyl, propyl,isopropyl, isobutyl, butyl, tert-butyl, sec-butyl, pentyl, and hexyl.

[0034] Preferred alkyl groups are C₁-C₈ alkyl.

[0035] “Alkenyl” means a carbon chain having one or two points ofunsaturation in the form of double bonds. Examples include ethenyl,prop-2-enyl, and hex-2,4-dienyl.

[0036] “Alkynyl” means a carbon chain having one or two triple bonds,for example, 2-butynyl, octa-3,5-diynyl, and the like.

[0037] The term “alkoxy” means an alkyl group such as C₁-C₈ alkylattached to an oxygen atom. Representative examples of alkoxy groupsinclude methoxy, ethoxy, tert-butoxy, propoxy, and isobutoxy.

[0038] The term “halogen” includes chlorine, fluorine, bromine, andiodine.

[0039] The foregoing alkyl, alkenyl, alkynyl, and alkoxy groups can besubstituted.

[0040] The term “substituted” means that one or more hydrogen atoms in amolecule has been replaced with another atom or group of atoms. Forexample, substituents include halogen, —OH, —CF₃, —NO₂, —NH₂,—NH(C₁-C₈alkyl), —N(C₁-C₈alkyl)₂, C₁-C₈ alkyl, —OC₁-C₈ alkyl, —CN, —CF₃,—CO₂H, —CO₂C₁-C₈ alkyl, SO₂H, and SO₂C₁-C₈ alkyl.

[0041] The term “substituted phenyl” means a phenyl ring in which from 1to 4 hydrogen atoms have been independently replaced with a substituent,preferably one selected from the list above. Examples of substitutedphenyl include 2,6-dichlorophenyl, 2-methoxycarbonylphenyl,3-cyanophenyl, 2,3,4,5-tetrafluorophenyl, 3-aminophenyl,2-hydroxyphenyl, and the like.

[0042] The symbol “—” means a covalent bond.

[0043] The term “pharmaceutically acceptable salt, ester, amide, andprodrug” as used herein refers to those carboxylate salts, amino acidaddition salts, esters, amides, and prodrugs of the compounds of thepresent invention which are, within the scope of sound medicaljudgement, suitable for use in contact with the tissues of patientswithout undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the invention. The term “salts” refers to therelatively nontoxic, inorganic and organic acid addition salts ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of the compounds or byseparately reacting the purified compound in its free base form with asuitable organic or inorganic acid and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laureate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactiobionate and laurylsulphonate salts, and the like.These may include cations based on the alkali and alkaline earth metals,such as sodium, lithium, potassium, calcium, magnesium, and the like, aswell as, nontoxic ammonium, quaternary ammonium and amine cationsincluding, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. (See, for example, Berge S. M.,et al., Pharmaceutical Salts, J. Pharm. Sci., 66:1-19 (1977) which isincorporated herein by reference.)

[0044] Examples of pharmaceutically acceptable, nontoxic esters of thecompounds of this invention include C₁-C₈ alkyl esters wherein the alkylgroup is a straight or branched chain. Acceptable esters also includeC₅-C₇ cycloalkyl esters as well as arylalkyl esters such as, but notlimited to benzyl. C₁-C₄ alkyl esters are preferred. Esters of thecompounds of the present invention may be prepared according toconventional methods.

[0045] Examples of pharmaceutically acceptable, nontoxic amides of thecompounds of this invention include amides derived from ammonia, primaryC₁-C₈ alkyl amines, and secondary C₁-C₈ dialkyl amines wherein the alkylgroups are straight or branched chain. In the case of secondary amines,the amine may also be in the form of a 5- or 6-membered heterocyclecontaining one nitrogen atom. Amides derived from ammonia, C₁-C₃ alkylprimary amides, and C₁-C₂ dialkyl secondary amides are preferred. Amidesof the compounds of the invention may be prepared according toconventional methods.

[0046] The term “prodrug” refers to compounds that are rapidlytransformed in vivo to yield the parent compound of the above formulas,for example, by hydrolysis in blood. A thorough discussion is providedin T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol.14 of the A. C. S. Symposium Series, and in Bioreversible Carriers inDrug Design, ed. Edward B. Roche, American Pharmaceutical Associationand Pergamon Press, 1987, both of which are incorporated herein byreference.

[0047] In addition, the compounds of the present invention can exist inunsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. In general, the solvatedforms are considered equivalent to the unsolvated forms for the purposesof the present invention.

[0048] The compounds of the present invention can exist in differentstereoisometric forms by virtue of the presence of asymmetric centers inthe compounds. It is contemplated that all stereoisometric forms of thecompounds, as well as mixture thereof, including racemic mixtures, formpart of this invention.

[0049] In the first step of the present method of imaging, a labeledcompound of Formula I is introduced into a tissue or a patient in adetectable quantity. The compound is typically part of a pharmaceuticalcomposition and is administered to the tissue or the patient by methodswell-known to those skilled in the art.

[0050] In the methods of the present invention, a compound can beadministered either orally, rectally, parenterally (intravenous, byintramuscularly or subcutaneously), intracisternally, intravaginally,intraperitoneally, intravesically, locally (powders, ointments ordrops), or as a buccal or nasal spray.

[0051] Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents,solvents, or vehicles include water, ethanol, polyols (propyleneglycol,polyethyleneglycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil), and injectable organic esters suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions and by the use of surfactants.

[0052] These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample sugars, sodium chloride, and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

[0053] Solid dosage forms for oral administration include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound is admixed with at least one inert customary excipient(or carrier) such as sodium citrate or dicalcium phosphate or (a)fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol, and silicic acid; (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia; (c) humectants, as for example, glycerol; (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates andsodium carbonate; (e) solution retarders, as for example paraffin; (f)absorption accelerators, as for example, quaternary ammonium compounds;(g) wetting agents, as for example, cetyl alcohol and glycerolmonostearate; (h) adsorbents, as for example, kaolin and bentonite; and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

[0054] Solid compositions of a similar type may also be employed asfillers in soft- and hard-filled gelatin capsules using such excipientsas lactose or milk sugar, as well as high molecular weightpolyethyleneglycols, and the like.

[0055] Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions which can be used arepolymeric substances and waxes. The active compounds can also be inmicroencapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

[0056] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, as for example,ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil, sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid estersof sorbitan or mixtures of these substances, and the like.

[0057] Besides such inert diluents, the composition can also includeadjuvants, such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and perfuming agents.

[0058] Suspensions, in addition to the active compounds, may containsuspending agents, as for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances, and the like.

[0059] Compositions for rectal administrations are preferablysuppositories which can be prepared by mixing the compounds of thepresent invention with suitable nonirritating excipients or carrierssuch as cocoa butter, polyethyleneglycol, or a suppository wax, whichare solid at ordinary temperatures but liquid at body temperature andtherefore, melt in the rectum or vaginal cavity and release the activecomponent.

[0060] Dosage forms for topical administration of a compound of thisinvention include ointments, powders, sprays, and inhalants. The activecomponent is admixed under sterile conditions with a physiologicallyacceptable carrier and any preservatives, buffers or propellants as maybe required. Ophthalmic formulations, eye ointments, powders, andsolutions are also contemplated as being within the scope of thisinvention.

[0061] In a preferred embodiment of the invention, the labeled compoundis introduced into a patient in a detectable quantity and aftersufficient time has passed for the compound to become associated withamyloid deposits, the labeled compound is detected noninvasively insidethe patient. In another embodiment of the invention, a labeled compoundof Formula I is introduced into a patient, sufficient time is allowedfor the compound to become associated with amyloid deposits, and then asample of tissue from the patient is removed and the labeled compound inthe tissue is detected apart from the patient. In a third embodiment ofthe invention, a tissue sample is removed from a patient and a labeledcompound of Formula I is introduced into the tissue sample. After asufficient amount of time for the compound to become bound to amyloiddeposits, the compound is detected.

[0062] The administration of the labeled compound to a patient can be bya general or local administration route. For example, the labeledcompound may be administered to the patient such that it is deliveredthroughout the body. Alternatively, the labeled compound can beadministered to a specific organ or tissue of interest. For example, itis desirable to locate and quantitate amyloid deposits in the brain inorder to diagnose or track the progress of Alzheimer's disease in apatient.

[0063] The term “tissue” means a part of a patient's body. Examples oftissues include the brain, heart, liver, blood vessels, and arteries. Adetectable quantity is a quantity of labeled compound necessary to bedetected by the detection method chosen. The amount of a labeledcompound to be introduced into a patient in order to provide fordetection can readily be determined by those skilled in the art. Forexample, increasing amounts of the labeled compound can be given to apatient until the compound is detected by the detection method ofchoice. A label is introduced into the compounds to provide fordetection of the compounds.

[0064] The term “patient” means humans and other animals. Those skilledin the art are also familiar with determining the amount of timesufficient for a compound to become associated with amyloid deposits.The amount of time necessary can easily be determined by introducing adetectable amount of a labeled compound of Formula I into a patient andthen detecting the labeled compound at various times afteradministration.

[0065] The term “associated” means a chemical interaction between thelabeled compound and the amyloid deposit. Examples of associationsinclude covalent bonds, ionic bonds, hydrophilic-hydrophilicinteractions, hydrophobic-hydrophobic interactions, and complexes.

[0066] Those skilled in the art are familiar with the various ways todetect labeled compounds. For example, MRI, positron emission tomography(PET), or single photon emission computed tomography (SPECT) can be usedto detect radiolabeled compounds. The label that is introduced into thecompound will depend on the detection method desired. For example, ifPET is selected as a detection method, the compound must possess apositron-emitting atom, such as ¹¹C or ¹⁸F.

[0067] Another example of a suitable label in a compound of Formula I isan atom such as ¹³C, ¹⁵N, or ¹⁹F which can be detected using MRI whichis also sometimes called nuclear magnetic resonance (NMR). In addition,the labeled compounds of Formula I may also be detected by MRI usingparamagnetic contrast agents.

[0068] Another example of detection is electron paramagnetic resonance(EPR). In this case, EPR probes which are well-known in the art, such asnitroxides, can be used.

[0069] The imaging of amyloid deposits can also be carried outquantitatively so that the amount of amyloid deposits can be determined.

[0070] The present invention also provides a method of inhibiting theaggregation of amyloid proteins to form amyloid deposits, byadministering to a patient in need of inhibition of the aggregation ofamyloid protein an amyloid protein inhibiting amount of a compound ofFormula I. Those skilled in the art are readily able to determine anamyloid inhibiting amount by simply administering a compound of FormulaI to a patient in increasing amounts until the growth of amyloiddeposits is decreased or stopped. The rate of growth can be assessedusing imaging or by taking a tissue sample from a patient and observingthe amyloid deposits therein.

[0071] A patient in need of inhibition of the aggregation of amyloidproteins is a patient having a central or peripheral disease orcondition in which amyloid proteins aggregate. Examples of such diseasesand conditions include Mediterranean fever, Muckle-Wells syndrome,idiopathetic myeloma, amyloid polyneuropathy, amyloid cardiomyopathy,systemic senile amyloidosis, amyloid polyneuropathy, hereditary cerebralhemorrhage with amyloidosis, Alzheimer's disease, Down syndrome,Scrapie, Creutzfeldt-Jacob disease, Kuru, Gerstmann-Straussler-Scheinkersyndrome, medullary carcinoma of the thyroid, Isolated atrial amyloid,β₂-microglobulin amyloid in dialysis patients, inclusion body myositis,β₂-amyloid deposits in muscle wasting disease, Sickle Cell Anemia,Parkinson's disease, and Islets of Langerhans diabetes type 2insulinoma.

[0072] Also provided by the present invention are compounds of Formula Iwherein one or more atom in the compound has been replaced with aradioisotope. The radioisotope can be any radioisotope. However, ³H,¹²³I, ¹I, ¹³¹I, ¹¹C, and ¹⁸F are preferred. Those skilled in the art arefamiliar with the procedure used to introduce a radioisotope into acompound. For example, compounds of Formula I are made where a ¹²C atomis replaced by a ¹³C atom.

[0073] The compounds of the present invention can be administered to apatient at dosage levels in the range of about 0.1 to about 1,000 mg perday, which are “effective amounts” for inhibiting amyloid formation andtreating the above mentioned diseases, especially Alzheimer's disease.For a normal human adult having a body weight of about 70 kg, a dosagein the range of about 0.01 to about 100 mg per kilogram of body weightper day is sufficient. The specific dosage used, however, can vary. Forexample, the dosage can depend on a number of factors including therequirements of the patient, the severity of the condition beingtreated, and the pharmacological activity of the compound being used.The determination of optimum dosages for a particular patient iswell-known to those skilled in the art.

[0074] The compounds of Formula I can be prepared by any of severalprocesses, utilizing readily available starting materials and methodswell-known in organic chemistry. Scheme I below illustrates a typicalmethod for making starting materials and the final products of FormulaI. The invention compounds are generally prepared by reacting an aminoindane with a phenyl halide such as a phenyl iodide or phenyl bromide.

[0075] Scheme 1 starts with a 2-amino-5-nitro-indane, which can beprepared by reacting 2-amino-indane with nitric acid and sulfuric acid,generally in a solvent such as trifluoroacetic acid.

[0076] The 2-amino-5-nitro-indane is readily reduced to thecorresponding 2,5-diaminoindane, for instance by hydrogenation in thepresence of a catalyst such as Raney nickel. The 5-amino group is thenreadily phenylated by reaction with a R⁴ phenyl compound

[0077] bearing a good leaving group L, for example where L is halo suchas iodo. The reaction is carried out under standard palladium-mediatedcoupling conditions, such as in an organic solvent (e.g., toluene) andin the presence of a mild base (e.g., cesium carbonate). The product, a2-amino-5-phenylaminoindane, is further alkylated or phenylated at the2-amino position, again utilizing standard alkylation methods.

[0078] Schemes 2, 3, and 4 illustrate the initial alkylation orphenylation of the 2-amino group of a 2-amino-5-nitro-indane, followedby reduction of the nitro group and phenylation of the 5-amino group.All of these reactions are carried out under standard conditions, forexample in an unreactive organic solvent, in the presence of a mildbase, and generally at an elevated temperature of about 60° C. to about150° C. The products are readily isolated by simply removing thereaction solvent, for example by evaporation under reduced pressure, andthey can be purified if desired by standard methods such aschromatography, crystallization, distillation, and the like.

[0079] The following detailed examples further illustrate the synthesisof typical compounds having Formula 1. The examples are representativeonly, and are not intended to limit the invention in any respect. Allreferences, including patents, cited herein are incorporated byreference.

Preparation 1

[0080] 2-Amino-5-nitro-indane Sulfate

[0081] Trifluoroacetic acid (60 mL) was charged into a flask, cooled inan ice/water bath, and 2-aminoindane hydrochloride (10.08 g) was addedcautiously, followed by H₂SO₄ (6.0 mL) and HNO₃ (3.0 mL). The ice bathwas removed, and the mixture was allowed to warm to room temperature andstir for 2 hours. The mixture was then placed in an ice water bath forthe slow addition of diethyl ether (300 mL) over 35 minutes. The mixturewas stirred at room temperature overnight, then the solids were filteredand dried in vacuo to give 15.43 g of 2-amino-5-nitro-indane sulfate.

Preparation 2

[0082] 2-(4-Fluorobenzyl)amino-5-nitro-indane and2-[bis-(4-Fluorobenzyl)amino]-5-nitro-indane

[0083] 2-Amino-5-nitro-indane sulfate (3 g) from Preparation I wasdissolved in 10 mL water and 20 mL 2 M NaOH. The mixture was extractedthree times with 40 mL methyl t-butyl ether, and the organic layers werecombined and dried over MgSO₄/Na₂SO₄, filtered, and concentrated byrotary evaporation. The residual oil was taken up in 30 mL CH₃CN, towhich 4-fluorobenzyl bromide (2.26 g) was added, followed by K₂CO₃. Themixture was heated at reflux for 18 hours, cooled to room temperature,filtered, and concentrated. The residual oil was purified by mediumpressure liquid column chromatography on silica gel (MPLC, biotagecolumn, solvent gradient 99:1 to 85:15 (CH₂Cl₂/1% NH₄OH in MeOH). Thedesired mono-alkylated aminoindane was obtained in 35% yield (1.10 g),and the bis-dialkylated aminoindane was also isolated (1.38 g, 32%yield). MS (APCI) m/z 287 (M⁺+1) monoalkylated, MS (APCI) m/z 395 (M⁺+1)dialkylated.

Preparation 3

[0084] General Conditions for Reduction of the Nitro Group

[0085] The nitro substituted indanes from Preparation 2 were reactedwith hydrogen in the presence of Raney nickel to give the corresponding2-alkyl and 2-dialkylamino-5-aminoindanes. The 2-amino-5-nitro-indanesulfate from Preparation I can be similarly hydrogenated to provide2,5-diamino-indane.

Preparation 4

[0086] 2-n-Pentylamino-5-nitro-indane and2-N,N-di-n-Pentylamino-5-nitro-indane

[0087] 2-Amino-5-nitro-indane sulfate from Preparation 1 (2 g) wasdissolved in 20 mL water and brought to pH=11 with 2N NaOH. The aqueoussolution was extracted with 3×30 mL methyl t-butyl ether, the organicswere combined, dried over MgSO₄/Na₂SO₄, filtered, and concentrated. Theresidual oil was dissolved in 30 mL acetonitrile, potassium carbonate (1g) and 1-bromopentane (1.79 mL) were added. The mixture was stirred atreflux for 18 hours, then filtered, concentrated, and purified by columnchromatography (MPLC, silica, gradient 1:1 to 3:1 ethyl acetate inhexanes) to give the mono-alkylated amine (0.26 g, 14% yield) and thedialkylated amine (0.99 g, 43% yield). MS mono-alkylated (APCI) m/z249.1 (M⁺+1). MS dialkylated (APCI) m/z 319.2 (M⁺+1).

Preparation 5

[0088] 2-(3,4-Dichlorobenzyl)amino-5-nitro-indane

[0089] Sulfuric acid (10 mL) was charged into a flask and cooled in anice/water bath containing 2-amino-5-nitro-indane hydrochloride (1.68 g),followed by HNO₃ (0.39 mL). The cooling bath was removed, and themixture was stirred at room temperature for 30 minutes, and then againcooled in an ice/water bath. Sodium hydroxide (25% soln) was addedcarefully to pH=11. The solution was then extracted with 4×60 mL diethylether. The combined organic layers were filtered through celite, driedover MgSO₄/Na₂SO₄, filtered, and concentrated. The residual black oilwas dissolved in THF (25 mL), cooled to 0° C. for the addition of NaH(0.4 g, 60% dispersion in mineral oil). After 5 minutes, dichloro benzylbromide (3.0 g) was added, and the reaction was stirred at roomtemperature for 16 hours. The reaction mixture was diluted with 20 mLwater and 40 mL Et₂O, the layers were separated, and the organics werewashed with 20 mL water. The aqueous layers were combined and washedwith 30 mL Et₂O, and the organic layers were combined, dried overMgSO₄/Na₂SO₄, filtered through celite, and concentrated. The black oilwas purified by column chromatography (MPLC, Isco ReadiSep silicacolumn, solvent gradient 80:20 to 50:50 Hexane/EtOAc) to give2-(3,4-dichlorobenzyl)amino-5-nitro-indane (1.05 g, 32% yield). MS(APCI) m/z 336.1 (M⁺−1). The nitro indane was reduced by reaction withhydrogen and Raney nickel to give2-(3,4-dichlorobenzyl)amino-5-amino-indane.

Preparation 6

[0090] 2-Amino-5-(3,4-dichlorophenylamino)-indane

[0091] 2,5-Diamino-indane (0.77 g) (from Preparation 3) was taken up intoluene (0.25 M), and nitrogen was bubbled through the solution for 5minutes. (S)-(−)-2,2′-bis(di-p-tolyl-phosphino)-1,1′-binaphthyl[(S)-tol-BINAP] (0.176 g) and palladium dibenzylidene acetone(Pd₂(dba)₃) (0.123 g), Cs₂CO₃ (2.37 g) and 3,4-dichloro-1-iodo benzene(1.42 g) was added, and the mixture was heated at reflux for 36 hours.The mixture was then cooled, diluted with diethyl ether, filteredthrough a plug of celite, concentrated, and purified by medium pressureliquid chromatography (silica gel column, eluted with CH₂Cl₂/MeOHgradient 1% MeOH to 20% MeOH). The fractions containing any trace of thedesired product (mass spec) were combined and concentrated to give2-amino-5-(3,4-dichlorophenylamino)-indane (0.54 g, 35% yield). MS(APCI) m/z 293.1 (M⁺+1).

EXAMPLE 1

[0092] 2-[(2-N,N-di-n-pentylamino)-indan-5-yl]amino-5-nitro-benzoic Acid

[0093] 2-N,N-di-n-Pentylamino-5-amino-indane (prepared as described inPreparation 3) (0.88 g) was dissolved in toluene (0.25 M), and nitrogenwas bubbled through the solution for 5 minutes. (S)-tol-BINAP (0.16 g)and Pd₂(dba)₂ (0.073 g), Cs₂CO₃ (1.39 g), and methyl 1-bromo-4-nitrobenzoate (0.66 g) were added, and the mixture was heated at reflux for18 hours. The mixture was then cooled, diluted with diethyl ether,filtered through a plug of celite, concentrated, and purified by mediumpressure liquid chromatography (silica gel column, eluted withCH₂Cl₂/MeOH gradient 1% MeOH to 10% MeOH). The fractions containing anytrace of the desired product (mass spec) were combined and concentratedto give methyl2-[(2-N,N-di-n-pentylamino)-indane-5-yl]amino-5-nitro-benzoate (1.28 g,90% yield). MS (APCI) m/z 468.1 (M⁺+1). The methyl benzoate (0.5 g) wasdissolved in 20 mL 1:1 THF/MeOH; this solution was treated with 4 mL 1 MLiOH and stirred at room temperature for 24 hours. When the startingmaterial was no longer observed by mass spec, the mixture wasconcentrated, and the residual oil was purified by column chromatography(MPLC, silica, gradient 98:2 to 80:20)(CH₂Cl₂/MeOH+1% NH₄OH). Fractionscontaining the desired product were combined, concentrated, and dried invacuo overnight to give2-[(2-N,N-di-n-pentylamino)-indan-5-yl]amino-5-nitro-benzoic acid (0.240g, 49% yield). MS (APCI) m/z 454.2 (M⁺+1). CHN for (C₂₆H₃₅N₃O₄) calc: C68.85, H 7.78, N 9.26; found: C 69.37, H 7.49, N 8.91.

EXAMPLE 2

[0094] Methyl 2-[2-(3,4-Dichloro-benzylamino)-indan-5-ylamino]-benzoate

[0095] 2-(3,4-Dichloro-benzylamino)-5-amino-indane (1.08 g) wasdissolved in toluene (0.25 M), and nitrogen was bubbled through thesolution for 5 minutes. (S)-tol-BINAP (0.12 g) and Pd₂(dba)₂ (0.08 g),Cs₂CO₃ (1.6 g), and methyl 1-bromo benzoate (0.76 g) were added, and themixture was heated at reflux for 24 hours. The mixture was then cooled,diluted with diethyl ether, filtered through a plug of celite,concentrated, and purified by medium pressure liquid chromatography(silica gel column, eluted with CH₂Cl₂/MeOH gradient 1% MeOH to 10%MeOH). The fractions containing any trace of the desired product (massspec) were combined and concentrated to give methyl2-[2-(3,4-dichloro-benzylamino)-indan-5-ylamino]-benzoate (0.98 g, 63%yield). MS (APCI) m/z 441.2 (M⁺+1). CHN for (C₂₄H₂₂Cl₂N₂O₂.0.13CH₃OH)calc: C 65.05, H 5.09, N 6.29; found: C 64.72, H 4.96, N 6.35.

EXAMPLE 3

[0096] 2-[2-(3,4-Dichlorobenzylamino)-indane-5-ylamino]-benzoic Acid

[0097] Methyl 2-[2-(3,4-dichloro-benzylamino)-indan-5-ylamino]-benzoate(0.98 g) was dissolved in 20 mL 1:1 THF/MeOH, and this solution wastreated with 9 mL 50% NaOH and stirred at room temperature for 24 hours.When the starting material was no longer observed by mass spec, themixture was concentrated to remove the MeOH, extracted with diethylether, and the precipitate was collected by filtration and dried invacuo overnight to give2-[2-(3,4-dichlorobenzylamino)-indane-5-ylamino]-benzoic acid (0.09 g,10% yield). MS (APCI) m/z 427.1 (M++I). CHN for (C₂₃H₂₀Cl₂N₂O₂.1.85HCl)calc: C 55.83, H 4.45, N 5.66; found: C 55.45, H 4.22, N 5.50.

EXAMPLE 4

[0098] 2-[2-(3,4-Dichlorobenzylamino)-indan-5-ylamine]-5-nitro-benzoicAcid

[0099] By following the procedure of Example 2,2-(3,4-dichlorobenzylamino)-5-amino-indane was reacted with methyl2-bromo-5-nitro benzoate to give methyl2-[2-(3,4-dichlorobenzyl-amino)-indan-5-yl-amino]-5-nitro-benzoate (0.65g, 69% yield). MS (APCI) m/z 486.0 (M⁺+1).

[0100] The methyl benzoate (0.65 g) was dissolved in 10 mL 1:1 THF/MeOH,and this solution was treated with 2 mL 50% NaOH and 1 mL water andstirred at room temperature for 18 hours. When the starting material wasno longer observed by mass spec, the mixture was concentrated, thesolids were dissolved in ethyl acetate/water 1:1. The layers wereseparated, and the aqueous layer was extracted with 2×20 mL ethylacetate. The organic layers were combined and washed with 10 mL 50%saturated NaCl, dried over MgSO₄/Na₂SO₄, filtered, and concentrated. Theresidual oil was purified by column chromatography (MPLC, silica,gradient 98:2 to 80:20)(CH₂Cl₂/MeOH+1% NH₄OH). Fractions containing thedesired product were combined, concentrated, and dried in vacuoovernight to give2-[2-(3,4-dichlorobenzylamino)-indan-5-ylamino]-5-nitro-benzoic acid(0.38 g, 60% yield). MS (APCI) m/z 470.0 (M⁺−1). CHN for(C₂₃H₁₉Cl₂N₃O₄.CH₂Cl₂) calc: C 55.58, H 3.94, N 8.31; found: C 55.25, H3.88, N 8.32.

EXAMPLE 5

[0101] 2-[2-(3,4-Dichlorobenzylamino)-indan-5-ylamino]-5-methoxy-benzoicAcid

[0102] By following the general procedure of Example 2,2-(3,4-dichlorobenzylamino)-5-amino-indane was reacted with methyl2-bromo-5-methoxy benzoate to give methyl2-[2-(3,4-dichlorobenzylamino)-indan-5-ylamino]-5-methoxy-benzoate(0.130 g, 11% yield). MS (APCI) m/z 471.0 (M⁺+1).

[0103] The methyl benzoate (0.130 g) was taken up in 20 mL 1:1 THF/MeOHand was treated with 1 M LIOH (3 mL). The mixture was stirred at roomtemperature for 48 hours. The mixture was then concentrated, and theresidual oil was purified by chromatography (MPLC, silica, gradient 95:5to 75:25 (CH₂Cl₂/MeOH). Fractions containing the desired product werecombined, concentrated, and dried in vacuo overnight to give2-[2-(3,4-dichlorobenzylamino)-indan-5-ylamino]-5-methoxy-benzoic acid(0.03 g, 24% yield). MS (APCI) m/z 457.0 (M⁺+1). CHN for(C₂₄H₂₂Cl₂N₂O₃.0.6H₂O) calc: C 61.57, H 4.98, N 5.98; found: C 61.23, H4.88, N 5.69.

EXAMPLE 6

[0104] 2-(2-Dipentylamino-indan-5-yl-amino)-5-methyl-benzoic Acid

[0105] 2-(2-Dipentylamino)-5-amino-indane (0.5 g) was taken up in THF(8.7 mL) and cooled to −78° C. for the addition of LiHMDS (4.0 mL, 1 Min THF). 2-Fluoro-5-methyl benzoic acid (0.27 g) was added after 5minutes, and the cold bath was removed. The mixture was allowed to warmto room temperature and stir for 16 hours. The mixture was diluted withdiethyl ether and water. The layers were separated, and the aqueouslayer was extracted with 2×20 mL diethyl ether. The organic layers werecombined, dried over MgSO₄/Na₂SO₄, filtered, and concentrated. Theresidual oil was purified by column chromatography (MPLC, silica,gradient 95:5 to 75:25)(CH₂Cl₂/MeOH). Fractions containing the desiredproduct were combined, concentrated, and dried in vacuo overnight togive 2-(2-dipentylamino-indan-5-yl-amino)-5-methyl-benzoic acid (0.029g, 4% yield). MS (APCI) m/z 421.2 (M⁺+1).

EXAMPLE 7

[0106] 4-(2-Dipentylamino-indan-5-yl-amino)-3-nitro-benzoic Acid

[0107] 2-(2-Dipentylamino)-5-amino-indane (0.5 g) was taken up in THF(8.7 mL) and cooled to −78° C. for the addition of LiHMDS (4.0 mL, 1 Min THF). 4-Fluoro-3-nitro benzoic acid (0.32 g) was added after 5minutes, and the cold bath was removed. The mixture was allowed to warmto room temperature and stir for 16 hours. The mixture was diluted withdiethyl ether and water. The layers were separated, and the aqueouslayer was extracted with 2×20 mL diethyl ether. The organic layers werecombined, dried over MgSO₄/Na₂SO₄, filtered, and concentrated. Theresidual oil was purified by column chromatography (MPLC, silica,gradient 95:5 to 75:25)(CH₂Cl₂/MeOH). Fractions containing the desiredproduct were combined, concentrated, and dried in vacuo overnight togive 4-(2-dipentylamino-indan-5-yl-amino)-3-nitro-benzoic acid (0.300 g,38% yield). MS (APCI) m/z 454.1 (M⁺+1).

EXAMPLE 8

[0108] Methyl2-[5-(3,4-dichlorophenylamino)-indan-2-ylamino]-5-nitro-benzoate

[0109] 2-Amino-5-(3,4-dichlorophenylamino)-indane (0.20 g) was taken upin toluene (0.25 M), and nitrogen was bubbled through the solution for 5minutes. (S)-tol-BINAP (0.023 g) and Pd₂(dba)₂ (0.05 g), Cs₂CO₃ (0.31g), and methyl 2-bromo-4-nitro benzoate (0.18 g) was added, and themixture was heated at reflux for 18 hours. The mixture was then cooled,diluted with diethyl ether, filtered through a plug of celite,concentrated, and purified by medium pressure liquid chromatography(silica gel column, eluted with CH₂Cl₂/MeOH gradient 1% MeOH to 20%MeOH). The fractions containing any trace of the desired product (massspec) were combined and concentrated to give methyl2-[5-(3,4-dichlorophenylamino)-indan-2-ylamino]-5-nitro-benzoate (0.10g, 26% yield). MS (APCI) m/z 472.0 (M⁺+1).

EXAMPLE 9

[0110] 2-[5-(3,4-Dichlorophenylamino)-indan-2-ylamino]-5-nitro-benzoicAcid

[0111] By following the procedure of Example 4, methyl2-[5-(3,4-dichlorophenylamino)-indan-2-ylamino]-5-nitro-benzoate wasreacted with sodium hydroxide in THF/MeOH to give2-[5-(3,4-dichlorophenylamino)-indan-2-ylamino]-5-nitro-benzoic acid(0.017 g, 18% yield). MS (APCI) m/z 457.9 (M⁺+1).

EXAMPLE 10

[0112] 2-[2-(4-Fluorobenzylamino)-indan-5-ylamino]-5-nitro-benzoic Acid

[0113] 2-(4-Fluorobenzylamino)-5-amino-indane (0.90 g) was taken up intoluene (0.25 M), and nitrogen was bubbled through the solution for 5minutes. (S)-tol-BINAP (0.18 g) and Pd₂(dba)₂ (0.08 g), Cs₂CO₃ (1.59 g),and methyl 1-bromo-4-nitro benzoate (0.75 g) were added, and the mixturewas heated at reflux for 48 hours. The mixture was then cooled, dilutedwith diethyl ether, filtered through a plug of celite, concentrated, andpartially purified by medium pressure liquid chromatography (silica gelcolumn, eluted with CH₂Cl₂/MeOH gradient 1% MeOH to 10% MeOH). Thefractions containing any trace of the desired product (mass spec) werecombined and concentrated. The residual crude oil was dissolved in 10 mL1:1 THF:MeOH with 1 mL 2N NaOH, and the reaction mixture was stirred atroom temperature 4 hours. When the starting material was no longerobserved by mass spec, the mixture was concentrated, and the residualoil was purified by column chromatography (MPLC, silica, gradient 95:5to 75:25 (CH₂Cl₂/1% NH₄OH in MeOH). Fractions containing the desiredproduct were combined, concentrated, and dried in vacuo overnight togive 0.83 g of2-[2-(4-fluorobenzylamino)-indan-5-ylamino]-5-nitro-benzoic acid. MS(APCI) m/z 422.0 (M⁺+1). CHN for (C₂₃H₂₀F₁N₃O₄.0.68[H₂O]) calc: C 63.70,H 4.96, N 9.69; Found: C 63.32, H 4.69, N 9.65.

EXAMPLE 11

[0114] 2-{2-[bis-(4-Fluorobenzyl)amino]indan-5-ylamino}-5-nitro-benzoicAcid

[0115] 2[bis-(4-Fluorobenzyl)amino]-5-amino-indane (1.28 g) was taken upin toluene (0.25 M), and nitrogen was bubbled through the solution for 5minutes. (S)-tol-BINAP (0.17 g) and Pd₂(dba)₂ (0.12 g), Cs₂CO₃ (1.6 g),and methyl 1-bromo-4-nitro benzoate (0.76 g) were added, and the mixturewas heated at reflux for 48 hours. The mixture was then cooled, dilutedwith diethyl ether, filtered through a plug of celite, concentrated, andpartially purified by medium pressure liquid chromatography (silica gelcolumn, eluted with CH₂Cl₂/MeOH gradient 1% MeOH to 10% MeOH). Thefractions containing any trace of the desired product (mass spec) werecombined and concentrated to give 1.89 g of the coupled ester. Theresidual crude oil was dissolved in 30 mL THF, and 20 mL of thissolution was treated with 5 mL 2N NaOH and stirred at room temperaturefor 12 hours. When the starting material was no longer observed by massspec, the mixture was concentrated, and the residual oil was purified bycolumn chromatography (MPLC, silica, gradient 95:5 to 75:25 (CH₂Cl₂/1%NH₄OH in MeOH). Fractions containing the desired product were combined,concentrated, and dried in vacuo overnight to give 0.063 g2-{2-[bis-(4-fluorobenzyl)amino]indan-5-ylamino}-5-nitro-benzoic acidpure. MS (APCI) m/z 530.0 (M⁺+1). CHN for (C₃₀H₂₅F₂N₃O₄.CH₃OH) calc: C66.22, H 5.23, N 7.46; Found: C 66.56, H 5.26, N 6.97.

EXAMPLE 12

[0116] 2-[2-(n-Pentylamino)-indan-5-ylamino]-5-nitro-benzoic Acid

[0117] 2-n-Pentylamino-5-amino-indane (0.32 g) was taken up in toluene(0.2 M), and nitrogen was bubbled through the solution for 5 minutes.(S)-tol-BINAP (0.076 g) and Pd₂(dba)₂ (0.04 g), Cs₂CO₃ (0.68 g), andmethyl 1-bromo-4-nitro benzoate (0.32 g) were added, and the mixture washeated at reflux for 48 hours. The mixture was then cooled, diluted withdiethyl ether, filtered through a plug of celite, concentrated, andpartially purified by medium pressure liquid chromatography (silica gelcolumn, eluted with CH₂Cl₂/MeOH gradient 1% MeOH to 10% MeOH). Thefractions containing any trace of the desired product (mass spec) werecombined and concentrated to give 0.42 g of the coupled ester. Theresidual crude oil was dissolved in 16 mL 1:1 THF/MeOH; this solutionwas treated with 1 mL 2N NaOH and stirred at room temperature for 12hours. When the starting material was no longer observed by mass spec,the mixture was concentrated, and the residual oil was purified bycolumn chromatography (MPLC, silica, gradient 98:2 to 80:20(CH₂Cl₂/MeOH). Fractions containing the desired product were combined,concentrated, and dried in vacuo overnight to give2-[2-(n-pentylamino)-indan-5-ylamino]-5-nitro-benzoic acid (0.170 g, 30%yield). MS (APCI) m/z 382.0 (M⁺−1). CHN for (C₂₁H₂₅N₃O₄.0.35CH₂Cl₂)calc: C 62.06, H 6.27, N 10.17; found: C 61.67, H 6.34, N 10.15.

Biological Examples

[0118] Invention compounds of Formula I can be evaluated in severalstandard in vitro and in vivo assays which are well-established asindicative of clinical usefulness in treating Alzheimer's disease andother conditions associated with amyloid formation.

[0119] Amyloid Assays

[0120] BASSR (feta-Amyloid Self-Seeding Radioassay)

[0121] An assay for inhibitors of self-seeded amyloid fibril growth

[0122] Materials:

[0123] Stock Solutions:

[0124] Assay Buffer—50 nM sodium phosphate, pH 7.5, 100 mM NaCl, 0.02%NaN₃, 1 M urea (filter and store at 4° C.)

[0125] Soluble Aβ(1-40) peptide (Bachem, Torrance, Calif.)—2.2 mg/mL indeionized H₂O (stored in aliquots at −20° C., keep on ice when thawed)will self-seed after 1 week storage. Typically, the solution should bestored until no lag phase is seen in the assay.

[0126]¹²⁵I-labeled Aβ(1-40)—150 to 350K cpm/μL in 100% acetonitrile—0.1%trifluoroacetic acid (TFA)—1% β-mercaptoethanol (aliquots stored at −20°C.). ¹²⁵I-labeled Aβ(1-40) can be made in accordance with the procedureset forth by H. Levine, III in Neurobiol. Aging, 16:755 (1995), which ishereby incorporated by reference, or this reagent may be purchased fromAmersham, Arlington Heights, Ill.

[0127] Final assay conditions: 30 μM soluble Aβ(1-40) in deionized waterin assay buffer+20 to 50K cpm ¹²⁵I-labeled Aβ(1-40) per assay. Compoundto be tested is dissolved in dimethylsulfoxide (DMSO), typically 5 to 50mM stock, such that the final concentration of DMSO is <1% v/v in theassay.

[0128] Assay: Reaction mixture for 50 assays (on ice) is comprised of0.1 to 0.2 μL of ¹²⁵I-labeled A¹²⁵I-labeled Aβ(1-40)+1 μL of solubleAβ(1-40)+13.5 μL assay buffer per assay. The following are the amountsof the components of the reaction mixture sufficient for 50 assay wells.

[0129] 5 to 10 μL ¹²⁵I-labeled Aβ(1-40) dried down

[0130] 675 μL assay buffer

[0131] 50 μL soluble Aβ(1-40)

[0132] Assay Method

[0133] 1) Prepare reaction mixture above by mixing components andstoring on ice.

[0134] 2) Pipet 14.5 μL of reaction mixture into each of 50 wells on apolypropylene U-bottom 96-well microtiter plate on ice (Costar 3794).

[0135] 3) Add 1.7 μL of diluted compound to be tested to each well in acolumn of eight, including solvent control. Serial 3-fold dilutions from1 mM (100 μM final) in assay buffer-urea=7 dilutions+zero. Each 96-wellplate can therefore accommodate 11 samples+I Congo Red control (0.039-5μM final in 2-fold steps).

[0136] 4) Seal the plate with aluminum film (Beckman 538619) andincubate for 10 minutes on ice.

[0137] 5) Raise the temperature to 37° C. and incubate for 3 to 5 hours(depending on the lot of the peptide).

[0138] 6) Remove the aluminum film and add 200 μL/well of ice cold assaybuffer with urea, collecting the radiolabeled fibrils by vacuumfiltration through 0.2-μm pore size GVWP filters in 96-well plates(Millipore MAGV N22, Bedford, Mass.). Determine the radioactivity of thefilters using standard methods well-known to those skilled in the art.

[0139] BASST (Beta-Amyloid Self-Seeding, ThioflavinT)

[0140] An assay for inhibitors of self-seeded amyloid fibril growth

[0141] METHODS:

[0142] Materials:

[0143] Stock Solutions:

[0144] Assay Buffer—50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 0.02%NaN₃, 1 M urea (filter and store at 4° C.)

[0145] Soluble Aβ(1-40)—2.2 mg/mL in deionized H₂O (store in aliquots at−20° C., keep on ice when thawed) will self-seed after 1 week storage.Typically, the solution should be stored until no lag phase is seen inthe assay.

[0146] Final assay conditions: 30 μM soluble Aβ(1-40) in deionized waterin assay buffer. Compound to be tested is dissolved in DMSO, typically 5to 50 mM stock, such that the final concentration of DMSO is <1% v/v inthe assay.

[0147] Assay: Reaction mixture for 50 assays (on ice) comprised of 1 μLof soluble Aβ(1-40)+13.5 μL assay buffer per assay. The following arethe amounts of the components of the reaction mixture that result ineach of the 50 assay wells.

[0148] 50 μL soluble Aβ(1-40)

[0149] 675 μL assay buffer

[0150] Assay Method

[0151] 1) Prepare the reaction mix above by mixing the components andstoring on ice.

[0152] 2) Pipet 14.5 μL of reaction mixture into each of 50 wells of apolystyrene U-bottom 96-well microtiter plate (Coming 25881-96) on ice.

[0153] 3) Add 1.7 μL of diluted compound to be tested to each well in acolumn of eight, including solvent control. Serial 3-fold dilutions from1 mM (100 μM final) in assay buffer-urea=7 dilutions+zero. Each 96-wellplate can therefore accommodate 11 samples+1 Congo Red control (0.039-5μM final in 2-fold steps).

[0154] 4) Seal the plate with aluminum film and incubate for 10 minuteson ice.

[0155] 5) Raise the temperature to 37° C. and incubate for 3 to 5 hours(depends on the lot of the peptide).

[0156] 6) Remove the aluminum film and add 250 μL/well of 5 μMthioflavin T (ThT) [T-3516, Sigma-Aldrich] in 50 mM glycine-NaOH, pH8.5. Read fluorescence on a plate reader (ex=440 nm/20 nm; em=485 nm/20nm) within 5 minutes.

[0157] BAPA (Beta-Amyloid Peptide Aggregation)

[0158] This assay is used to provide a measure of inhibition by acompound against the aggregation behavior of the β-amyloid peptide.

[0159] The purpose of this assay is to provide a higher volume method ofassaying the amount of β-amyloid aggregation using an endpoint assaybased on filtration. In this assay, hexafluoroisopropanol (HFIP) is usedto break down the initial amyloid peptide to a monomer state and use aconcentration of 33 μM which is high enough so that aggregation willoccur at pH 6.0 in several hours.

[0160] METHODS:

[0161] Beta-Amyloid Peptide Aggregation, pH 6.0 (BAPA)

[0162] In a 96-well plate (Costar 3794), we add 25 μL 50 mM phosphatebuffer, pH 6.0, 10 μL 0.5 mg/mL Aβ (1-40) peptide in 20% HFIP+0.1μL/assay radioiodinated ¹²⁵I Aβ (1-40) [¹²⁵I Aβ(1-40)], and 1 μL of thecompound to be tested starting at 50 mM with a concentration of DMSO<1%.Then, we incubate for 2 to 4 hours at room temperature. We stop thereaction with 200 μL of 50 mM phosphate buffer, pH 6.0, and filter itthrough a 0.2 μm 96-well filter plate (Millipore MAGU N22). We wash thefilter plate with 100 μL of the same phosphate buffer. Aggregation wasdetected on a Microbeta counter after impregnating the filters withMeltilex (1450-441) and is corrected for background.

[0163] Batym Assay

[0164] METHODS:

[0165] Required Aβ (1-42) (California Peptide) was dried from its HFIPstock solution. The Aβ (1-42) was dissolved in DMSO and then mixed withphosphate buffered saline (PBS) (pH 7.4). The mixed Aβ (1-42) solutionwas filtered with a Omnipore membrane 0.2-μm syringe filter (Millipore,Bedford, Mass.). The compound to be tested in DMSO (50× concentrate) wasput into each well (0.5 μL/well) of a 96-well plate. The Aβ (1-42)solution was added into each well (24.5 μL/well). The plate wascentrifuged at 1,000 g for 5 minutes and incubated at 37° C. for 1 day(Aβ1-42; final concentration 100 μM).

[0166] After incubation, Thioflavin T (ThT) (30 μM) solution inglycine-NaOH buffer (pH 8.5, 50 mM) was added into each well (250μL/well), fluorescence was measured (ex=440/20 nm; em=485/20 nm) using afluorescence plate reader. The inhibitory activity was calculated as thereduction of fluorescence with the following formula:

Inhibition (%)={(F(Aβ)−F(Aβ+compound)}/{F(Aβ)−F(solvent+compound)}×100.

[0167] The IC₅₀s were calculated by a curve fitting program using thefollowing equation. The data were obtained from two differentexperiments in triplicate.

[0168] Inhibition (x)=100−100/{1+(x/IC₅₀)^(n)}.

[0169] x=concentration of tested compound (M).

[0170] IC₅₀=(M).

[0171] n=Hill coefficient.

[0172] Representative compounds of Formula I have exhibited inhibitoryactivities (IC₅₀) ranging from about 0.1 to>100 μM in the foregoingassays. The results of these assays for specific representativecompounds of the present invention are shown in the table below. AmyloidInhibition Example BASST BAYTM BASSR BAPA No. IC₅₀ μM IC₅₀ μM IC₅₀ μMIC₅₀ μM 1 40 >100 >100, >100, >100 33 3 40 >100 >100 4 1.116 >100, >100, 7.5, 8, 9 28 5 30 >100 >100, >100, 30 (ppt), 100 165 621, >100 >100 (2×) >100 (5×) 118 7 8 >100 >100, >100, 15, >100 118 90.25 >100, 8 (v), 7 (v), 6 (ppt) 5 10 30 53.8 >100, >100, 90, 10 >100 111 7, 12 >100, 20 (ppt), 1.2 (ppt), 7 >100 12 >100 >100 >100, >100 5

[0173] The invention compounds can also be evaluated in standard in vivoassays commonly used to evaluate agents to treat conditions associatedwith amyloid aggregation such as Alzheimer's disease. In one assay,amyloid protein is induced into the spleen of mice by subcutaneousinjections of silver nitrate, Freund's complete adjuvant, and anintravenous injection of amyloid enhancing factor. Silver nitrate isadministered each day through Day 11. Test compounds are administered tothe mice daily starting on Day 1 through Day 11. On Day 12, the animalsare sacrificed, and the spleens are removed, histologically prepared,stained with Congo red, and the percent area of the spleen occupied bybirefringent, Congo red-stained amyloid is quantitated microscopically.Invention compounds will inhibit splenic amyloid deposition.

[0174] Another in vivo assay in which the invention compounds can beevaluated uses transgenic mice. The mice bear a human β-amyloidprecursor protein transgene with a prion promoter, as descried by Hsiaoet al., “Correlative memory deficits, Aβ elevation, and amyloid plaquesin transgenic mice,” Science 1996;274:99-102. These transgenic micedevelop β-amyloid deposits at about 9 months of age. By 15 months,diffuse and compact senile plaques are abundant, primarily in theneocortex, olfactory bulb, and hippocampus. Invention compounds areadministered orally to the mice beginning at the age of 8 months (justprior to the onset of amyloid deposits) and continuing for severalmonths (up to about age 14-18 months). The animals are then sacrificed,and the brains are removed. The amount of amyloid in the brain isquantitated both histologically and biochemically. Invention compoundswill inhibit amyloid accumulation in the cortex and hippocampus of thetest animals.

[0175] The foregoing data establish that invention compounds of FormulaI are potent inhibitors of protein aggregation, and are thus useful intreating diseases associated with amyloid deposits and to image amyloiddeposits for diagnostic use. The compounds typically will be used in theform of pharmaceutical formulations for therapeutic use, and thefollowing examples further illustrate typical compositions. EXAMPLE 13Tablet Formulation Ingredient Amount Compound of Example 1 50 mg Lactose80 mg Cornstarch (for mix) 10 mg Cornstarch (for paste) 8 mg MagnesiumStearate (1%) 2 mg 150 mg

[0176] The compound of Example 1 is mixed with the lactose andcornstarch (for mix) and blended to uniformity to a powder. Thecornstarch (for paste) is suspended in 6 mL of water and heated withstirring to form a paste. The paste is added to the mixed powder, andthe mixture is granulated. The wet granules are passed through a No. 8hard screen and dried at 50° C. The mixture is lubricated with 1%magnesium stearate and compressed into a tablet. The tablets areadministered to a patient at the rate of 1 to 4 each day for preventionof amyloid and treatment of Alzheimer's disease.

EXAMPLE 14

[0177] Parenteral Solution

[0178] In a solution of 700 mL of propylene glycol and 200 mL of waterfor injection is added 20.0 g of compound of Example 9. The mixture isstirred, and the pH is adjusted to 5.5 with hydrochloric acid. Thevolume is adjusted to 1000 mL with water for injection. The solution issterilized, filled into 5.0 mL ampoules, each containing 2.0 mL (40 mgof compound of Example 9), and sealed under nitrogen. The 10 solution isadministered by injection to a patient suffering from medullarycarcinoma of the thyroid and in need of treatment.

EXAMPLE 15

[0179] Patch Formulation

[0180] Ten milligrams of2-[2-(3,4-dichlorobenzylamino)-indan-5-yl-amino]-benzoic acid is mixedwith 1 mL of propylene glycol and 2 mg of acrylic-based polymer adhesivecontaining a resinous cross-linking agent. The mixture is applied to animpermeable backing (30 cm²) and applied to the upper back of a patientfor sustained release treatment of amyloid polyneuropathy.

[0181] The invention and the manner and process of making and using itare now described in such full, clear, concise, and exact terms as toenable any person killed in the art to which it pertains, to make anduse the same. It is to be understood that the foregoing describespreferred embodiments of the present invention and that modificationsmay be made therein without departing from the spirit or scope of thepresent invention as set forth in the claims. To particularly point outand distinctly claim the subject matter regarded as invention, thefollowing claims conclude this specification.

What is claimed is:
 1. A compound of the Formula I

wherein: R¹ and R² independently are hydrogen, C₁-C₈ alkyl, C₂-C₈alkenyl, C₂-C₈ alkynyl, (CH₂)_(n) phenyl or (CH₂)_(n) substitutedphenyl, provided that one of R¹ and R² is other than hydrogen; R⁴ and R⁵independently are hydrogen, halo, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, (CH₂)_(n) phenyl, (CH₂)_(n) substituted phenyl, NO₂, CN, CF₃,C₁-C₈ alkoxy, CO₂R⁶, tetrazolyl, NH(C₁-C₈ alkyl), N(C₁-C₈ alkyl)₂, orSO₂R⁶; R³ is hydrogen or C₁-C₈ alkyl; R⁶ is hydrogen, C₁-C₈ alkyl, or(CH₂)_(n)phenyl or (CH₂)_(n) substituted phenyl; n is an integer from 0to 4 inclusive; or a pharmaceutically acceptable salt, ester, amide, orprodrug thereof.
 2. A compound of claim 1 having Formula II


3. A compound of claim 2 wherein R⁶ is hydrogen.
 4. A compound of claim1 having Formula III

wherein R⁷ is hydrogen, halo, NO₂, CN, C₁-C₈ alkyl, C₁-C₈ alkoxy, CF₃,NH₂, NH(C₁-C₈ alkyl), or N(C₁-C₈ alkyl)₂.
 5. A compound selected fromthe group consisting of2-[(2-N,N-di-n-pentylamino)-indan-5-yl]amino-5-nitro-benzoic acid;Methyl 2-[2-(3,4-Dichloro-benzylamino)-indan-5-ylamino]-benzoate;2-[2-(3,4-Dichlorobenzylamino)-indane-5-ylamino]-benzoic acid;2-[2-(3,4-Dichlorobenzylamino)-indan-5-ylamine]-5-nitro-benzoic acid;2-[2-(3,4-Dichlorobenzylamino)-indan-5-ylamino]-5-methoxy-benzoic acid;2-(2-Dipentylamino-indan-5-yl-amino)-5-methyl-benzoic acid;4-(2-Dipentylamino-indan-5-yl-amino)-3-nitro-benzoic acid; Methyl2-[5-(3,4-dichlorophenylamino)-indan-2-ylamino]-5-nitro-benzoate;2-[5-(3,4-Dichlorophenylamino)-indan-2-ylamino]-5-nitro-benzoic acid;2-[2-(4-Fluorobenzylamino)-indan-5-ylamino]-5-nitro-benzoic acid;2-{2-[bis-(4-Fluorobenzyl)amino]indan-5-ylamino}-5-nitro-benzoic acid;and 2-[2-(n-Pentylamino)-indan-5-ylamino]-5-nitro-benzoic acid.
 6. Amethod of treating Alzheimer's disease, the method comprisingadministering to a patient having Alzheimer's disease a therapeuticallyeffective amount of a compound of claim
 1. 7. A method of treatingAlzheimer's disease, the method comprising administering to a patienthaving Alzheimer's disease a therapeutically effective amount of acompound of claim
 2. 8. A method of inhibiting the aggregation ofamyloid proteins to form amyloid deposits, the method comprisingadministering to a patient in need of inhibition of amyloid proteinaggregation an amyloid protein aggregation inhibiting amount of acompound of claim
 1. 9. A method of inhibiting the aggregation ofamyloid proteins to form amyloid deposits, the method comprisingadministering to a patient in need of inhibition of amyloid proteinaggregation an amyloid protein aggregation inhibiting amount of acompound of claim
 2. 10. A method of inhibiting the aggregation ofamyloid proteins to form amyloid deposits, the method comprisingadministering to a patient in need of inhibition of amyloid proteinaggregation an amyloid protein aggregation inhibiting amount of acompound of claim
 3. 11. A method of imaging amyloid deposits, themethod comprising the steps of: a) introducing into a patient adetectable quantity of a labeled compound of claim 1; b) allowingsufficient time for the labeled compound to become associated withamyloid deposits; and c) detecting the labeled compound associated withthe amyloid deposits.
 12. A method of imaging amyloid deposits, themethod comprising the steps of: a) introducing into a patient adetectable quantity of a labeled compound of claim 2; b) allowingsufficient time for the labeled compound to become associated withamyloid deposits; and c) detecting the labeled compound associated withthe amyloid deposits.
 13. A method of imaging amyloid deposits, themethod comprising the steps of: a) introducing into a patient adetectable quantity of a labeled compound of claim 3; b) allowingsufficient time for the labeled compound to become associated withamyloid deposits; and c) detecting the labeled compound associated withthe amyloid deposits.
 14. The method of claim II wherein the patient hasor is suspected to have Alzheimer's disease.
 15. The method of claim 12wherein the patient has or is suspected to have Alzheimer's disease. 16.The method of claim 13 wherein the patient has or is suspected to haveAlzheimer's disease.
 17. The method of claim 11 wherein the labeledcompound is a radiolabeled compound.
 18. The method of claim 12 whereinthe labeled compound is a radiolabeled compound.
 19. The method of claim13 wherein the labeled compound is a radiolabeled compound.
 20. Themethod of claim 11 wherein the labeled compound is detected using MRI.21. The method of claim 12 wherein the labeled compound is detectedusing MRI.
 22. The method of claim 13 wherein the labeled compound isdetected using MRI.
 23. A pharmaceutical composition comprising acompound of claim 1 together with an excipient, diluent, or carriertherefor.
 24. A pharmaceutical composition comprising a compound ofclaim 2 together with an excipient, diluent, or carrier therefor.
 25. Apharmaceutical composition comprising a compound of claim 3 togetherwith an excipient, diluent, or carrier therefor.
 26. A pharmaceuticalcomposition comprising a compound of claim 4 together with an excipient,diluent, or carrier therefor.